bims-tricox 2025-02-16 papers

Issue of 2025–02–16
five papers selected by
Yash Verma, University of Zurich

Cell Rep. 2025 Jan 28. pii: S2211-1247(24)01494-3. [Epub ahead of print]44(1): 115143

A microscopy-based screen identifies cellular kinases modulating mitochondrial translation.

Roya Yousefi, Luis Daniel Cruz-Zaragoza, Anusha Valpadashi, Carina Hansohn, Drishan Dahal, Ricarda Richter-Dennerlein, Silvio Rizzoli, Henning Urlaub, Peter Rehling, David Pacheu-Grau.

Mitochondrial DNA encodes 13 subunits of the oxidative phosphorylation (OXPHOS) system, which are synthesized inside the organelle and essential for cellular energy supply. How mitochondrial gene expression is regulated and integrated into cellular physiology is little understood. Here, we perform a high-throughput screen combining fluorescent labeling of mitochondrial translation products with small interfering RNA (siRNA)-mediated knockdown to identify cellular kinases regulating translation. As proof of principle, the screen identifies known kinases that affect mitochondrial translation, and it also reveals several kinases not yet linked to this process. Among the latter, we focus on the primarily cytosolic kinase, fructosamine 3 kinase (FN3K), which localizes partially to the mitochondria to support translation. FN3K interacts with the mitochondrial ribosome and modulates its assembly, thereby affecting translation. Overall, our work provides a reliable approach to identify protein functions for mitochondrial gene expression in a high-throughput manner.

Keywords: CP: Metabolism; CP: Molecular biology; cellular kinases; click chemistry; mito-FUNCAT; mitochondrial translation; siRNA library

DOI: https://doi.org/10.1016/j.celrep.2024.115143

FEBS J. 2025 Feb 11.

Saccharomyces cerevisiae Dmo2p is required for the stability and maturation of newly translated Cox2p.

Maria Antônia Kfouri Martins Soares, Letícia Veloso Ribeiro Franco, Jhulia Almeida Clarck Chagas, Fernando Gomes, Mário H Barros.

Based on available platforms detailing the Saccharomyces cerevisiae mitochondrial proteome and other high-throughput studies, we identified the yeast gene DMO2 as having a profile of genetic and physical interactions that indicate a putative role in mitochondrial respiration. Dmo2p is a homologue to human distal membrane-arm assembly complex protein 1 (DMAC1); both proteins have two conserved cysteines in a Cx2C motif. Here, we localised Dmo2p in the mitochondrial inner membrane with the conserved cysteines facing the intermembrane space. The respiratory deficiency of dmo2 mutants at 37°C led to a reduction in cytochrome c oxidase (COX) activity (COX) and in the formation of cytochrome bc1 complex-COX supercomplexes; dmo2 also has a rapid turnover of Cox2p, the second subunit of the COX complex that harbours the binuclear CuA centre. Moreover, Dmo2p co-immunoprecipitates with Cox2p and components required for maturation of the CuA centre, such as Sco1p and Sco2p. Finally, DMO2 overexpression can suppress cox23 respiratory deficiency, a mutant that has impaired mitochondrial copper homeostasis. Mass spectrometry data unveiled the interaction of Dmo2p with different large molecular complexes, including bc1-COX supercomplexes, the TIM23 machinery and the ADP/ATP nucleotide translocator. Overall, our data suggest that Dmo2p is required for Cox2p maturation, potentially by aiding proteins involved in copper transport and incorporation into Cox2p.

Keywords: COX assembly; CuA site formation; DMAC1 homologue

DOI: https://doi.org/10.1111/febs.70009

Arch Biochem Biophys. 2025 Feb 07. pii: S0003-9861(25)00042-6. [Epub ahead of print]766 110329

Annealing synchronizes the TOM complex with Tom7 in a new orientation.

Liuyan Yang, Mingdong Liu, Lei Qi, Yunhui Liu, Xubo Lin, Yu-Zhong Zhang, Qing-Tao Shen.

Annealing is an ideal approach to synchronizing soluble proteins into their minimum-energy states via tandem heating and cooling treatments. Like soluble proteins, many membrane proteins also suffer intrinsic structural flexibility, the major obstacle to high-resolution structural determination. How to apply annealing onto membrane proteins remains unexplored. Here, we utilized the translocase of the outer mitochondrial membrane (TOM) as the model and investigated the ideal annealing conditions for membrane proteins. After structural determination via cryo-electron microscopy, we indicated that fast cooling the heated TOM complex to 0 °C can significantly improve the local resolution compared with the unannealed one. Structural analyses showed that annealing renders the TOM complex into a new conformation with its Tom7 α1 helix from a reclining position on the membrane surface to a lying orientation, accompanied by the loop between β6 and β7 in Tom40, flipping outward from the Tom40 β-barrel, ideal for preprotein translocation. In all, our results demonstrate the role of annealing in synchronizing membrane proteins and unveil unidentified conformations of the TOM complex.

Keywords: Annealing; Membrane proteins; The TOM complex

DOI: https://doi.org/10.1016/j.abb.2025.110329

Mol Cell. 2025 Jan 30. pii: S1097-2765(25)00052-8. [Epub ahead of print]

The RNA helicase HrpA rescues collided ribosomes in E. coli.

Annabelle Campbell, Hanna F Esser, A Maxwell Burroughs, Otto Berninghausen, L Aravind, Thomas Becker, Rachel Green, Roland Beckmann, Allen R Buskirk.

Although many antibiotics inhibit bacterial ribosomes, the loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in E. coli, suggesting the existence of additional mechanisms. Here, we show that the RNA helicase HrpA rescues stalled ribosomes in E. coli. Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis to split stalled ribosomes into subunits. Cryoelectron microscopy (cryo-EM) structures reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA such that, by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome. These studies show that ribosome splitting is a conserved mechanism that allows proteobacteria to tolerate ribosome-targeting antibiotics.

Keywords: DEAH-box protein; HrpA; SmrB; cryo-EM; ribosome collisions; ribosome splitting

DOI: https://doi.org/10.1016/j.molcel.2025.01.018

Mol Cell. 2025 Feb 06. pii: S1097-2765(25)00048-6. [Epub ahead of print]

RIOK3 mediates the degradation of 40S ribosomes.

Zixuan Huang, Frances F Diehl, Mengjiao Wang, Yi Li, Aixia Song, Fei Xavier Chen, Nicolle A Rosa-Mercado, Roland Beckmann, Rachel Green, Jingdong Cheng.

Cells tightly regulate ribosome homeostasis to adapt to changing environments. Ribosomes are degraded during stress, but the mechanisms responsible remain unclear. Here, we show that starvation induces the selective depletion of 40S ribosomes following their ubiquitylation by the E3 ligase RNF10. The atypical kinase RIOK3 specifically recognizes these ubiquitylated 40S ribosomes through a unique ubiquitin-interacting motif, visualized by cryoelectron microscopy (cryo-EM). RIOK3 binding and ubiquitin recognition are essential for 40S ribosome degradation during starvation. RIOK3 induces the degradation of ubiquitylated 40S ribosomes through progressive decay of their 18S rRNA beginning at the 3' end, as revealed by cryo-EM structures of degradation intermediates. Together, these data define a pathway and mechanism for stress-induced degradation of 40S ribosomes, directly connecting ubiquitylation to regulation of ribosome homeostasis.

Keywords: 40S; RIOK3; RNA degradation; cryo-EM; homeostasis; ribosome; starvation; stress response; translation; ubiquitin

DOI: https://doi.org/10.1016/j.molcel.2025.01.013